Spiral ganglion neurons (SGN) replacement is critical for the treatment of sensorineural hearing loss using a stem cell (SC)-based strategy. However, this repair strategy is unlikely to restore hearing if neural connections are not established between the implanted cells and the brain. This proposal will investigate the hypothesis that peripherally implanted neurons can form ascending neurites and make synaptic contacts with cochlear nucleus (CN). This hypothesis is supported by previous observations that embryonic SC (ESC)-derived neurons can invade brain slices and form synapses with brain neurons. A recent study in our lab found that NTs stimulated neurite outgrowth from cochlear-vestibular ganglion-neural SC-derived neurons (CVG-NSC-neurons) in vitro and that in vivo-implanted CVG-NSC-neurons extended neurites along the cranial nerve VIII (VIII nerve) tract toward the CN. These findings suggest that the reconstruction of neural circuitry between the SGN area and CN is possible. The central objective of this proposal is to develop strategies to stimulate exogenous neurons to generate neurites along the VIII nerve that form synaptic contacts with CN neurons following treatment with NT. To achieve this goal, we propose the following specific aims: (1) Determine whether NTs induce glutamatergic neuronal differentiation of CVG-NSCs. (2) Explore whether NTs stimulate exogenous neurons to extend neurite outgrowth toward the CN and make synapse-like contacts using a cochlea-VIII nerve-CN explant model. (3) Assess the ability of exogenous neurons to extend neurites toward the CN and form synapse-like contacts in an in vivo model. This proposal will explore the integration of exogenous neurons into the host central auditory system, a critical step in SC-based hearing regeneration. The results will provide insights into reconstructing the ascending neural pathway from the peripheral inner ear to the CN. The development of an animal model will lead to research toward potential applications in humans. These findings may be used to improve the efficacy of cochlear implants, which rely on SGN stimulation to communicate auditory information to the brain. 1 PUBLIC HEALTH RELEVANCE: A stem cell-based replacement has been proposed to substitute damaged spiral ganglion neurons in hearing loss. This proposal investigates whether exogenous neurons can rebuild the neural circuits from the peripheral auditory system to the central auditory system. The development of an animal model will lead to research toward potential applications in humans. The success of this proposal will provide insights into the ascending auditory pathway reconstruction in the replacement studies for hearing loss, tinnitus, vestibular disorders, and other neurodegenerative diseases.